DESCRIPTION: Dr. Quake proposes to develop a new method and instrument for sequencing that will increase both the length of the read frame and the throughput for large scale sequencing. The instrument combines standard microfabrication techniques with optoelectronic techniques for single molecule detection. The sequencing scheme is summarized as follows. A microfabricated flow device will be constructed, in which various reagents can be flowed through a chamber of linear dimension 10um-100um. The chamber will have a window to allow optical interrogation. Single stranded DNA molecules with primers will be anchored to the surface of the chamber, with streptavidin-biotin links. Then DNA polymerase and one of the four nucleotide triphosphates (with some fraction fluorescently labeled) will be flowed into the chamber, incubated with the DNA, and flowed out. If the labeled nucleotide is incorporated, a fluorescent signal will be detected. If no signal is detected, the process is repeated with a different nucleotide. Once the signal is detected, the fluorophores will be excited until they photobleach. This prevents previously detected bases from interfering with the current base. This scheme can be iterated ad infinitum, and a read length of at least 3kbp is anticipated. Since the scheme depends crucially on the ability to photobleach the signal before incorporating the next base, it is called fluorescent photobleaching sequencing (FPS). Specifically, Dr. Quake proposes to 1 ) Build a prototype device to implement this sequencing scheme with a microfabricated flow cell and an external electronic valve to control fluid flow. 2) Demonstrate sequencing with the prototype by sequencing all or some fraction of a 3 kbp insert in a single stranded M13 vector. 3) Test the efficiency, throughput and read length of the device. These parameters will be optimized by varying the fraction of labeled nucleotides, the buffer conditions, the number of bound DNA molecules, etc. 4) Build a second generation device with 10um channel depths and integrated microfabricated valves. 5) Show that the second generation device has vastly improved throughput and efficiency for sequencing. 6) Design and fabricate a parallel layout with several devices on a single silicon wafer.